1. Field of the Invention
This invention relates to a growth supplement for culture medium for the maintenance of cell viability and for the induction of differentiation, especially of bacterial cells. It also relates to a method for growing cells, especially enteric bacteria such as variants of Salmonella enterica, for the production of biologics, such as vaccines, assays and the enhancement of protein secretion in general.
2. Description of the Prior Art
A medium which induces cell differentiation in both prokaryote and eukaryote cells will aid in understanding neoplasia, birth defects, regeneration of nervous system function after debilitating accidents, development of artificial models for diseases such as Alzheimer's disease and diabetes, as well as the production of improved vaccines to infectious disease agents and identification of contamination of animal products that are a part of the human food supply.
For example, rapid and reliable detection of Salmonella serotypes is important for determining the presence of Salmonella in food and animals, such as for example, chickens used for egg production. Standard methods for Salmonella detection are often not sensitive enough to consistently detect the presence of Salmonella serotypes in all instances. Fowl salmonellosis caused by Salmonella enterica var. Gallinarium and Pullorum are diseases of world wide significance in the poultry industry. Since its recognition as an infectious agent nearly 100 years ago, Salmonella enterica var. Pullorum has been classified as a non-motile, aflagellate chicken pathogen (Retter, N.Y. Med. J., Volume 71, 803,1990; Edwards et al, Identification of Enterobacteriaceae, Burgess Publishing Company, Minneapolis, Minn., 1962). It is particularly capable of contaminating eggs via the reproductive organs of hen (Snoeyenbos, Diseases of Poultry, ninth edition; Ed.: B. W. Calnek; 73-86, 1991). In the United States, S. enterica var. Pullorum was a major problem in the poultry industry earlier this century. The problem decreased following implementation of rigorous serological screening and depopulation of serologically positive flocks. Serological surveillance has been the primary method of keeping S. enterica var. Pullorum in check. However, problems have begun to erupt in a number of United States flocks and indications are that the standard tests for S. enterica var. Pullorum, such as the agglutination assays, are not detecting some infected flocks. Currently, S. enterica var. Enteritidis is the cause of a world-wide increase of salmonellosis in people due to its ability to colonize the reproductive organs of chickens and to contaminate eggs. S. enterica var. Enteritidis has the same lipopolysaccharide (LPS) D1 serotype as S. enterica var. Pullorum, but it is commonly flagellated and produces flagellar H-antigens that are used in a diagnostic scheme to differentiate it from other salmonellae (Edwards, supra). Glycosylated, high-molecular weight (HMW) O-antigen distinguishes virulent strains (Petter, Appl. Env. Microbiol., Volume 59, 2884-2890, 1993; Guard-Petter et al, Appl. Env, Microbiol., Volume 61, 2845-2851, 1995; Guard-Petter et al, Epid. Infec., 1996, In Press; R. Carlson, personal communication). This type of O-antigen structure contributes to the ability of S. enterica var. Enteritidis to hyperflagellate and migrate across a 2% agar surface, but this distinctive phenotype is transient and is lost upon passage and storage (Guard-Petter et al, 1995, supra; Guard-Petter et al, 1996, supra). Hyperflagellation has been described as an outer membrane change that occurs with the differentiation of vegetative bacteria into swarm cells (Allison et al, Molec. Microbiol., Volume 169, 1155-1158, 1994; Allison et al, Infec. Immun., Volume 60, 4740-4746, 1992). A genetic analysis of E. coli and S. enterica var. Typhimurium indicated that swarm cell differentiation could be observed in soft agar (Harshay et al, PNAS, USA, Volume 91, 8631-8635, 1994). A correlation between differentiation and virulence has been described for the urinary tract pathogen Proteus mirabilis (Allison et al, 1994, supra; Allison et al, 1992, supra) where other virulence factors such as toxins and metalloproteases are transcriptionally upregulated at the same time as flagellin. To date, a relationship between virulence and hyperflagellation for S. enterica var. Enteritidis has been made only in those strains that swarm on 2% agar surfaces, because even avirulent rough and semismooth strains of S. enterica var. Enteritidis produce flagella and are motile in soft agar (Guard-Petter et al, 1996, supra). These emerging concepts suggest that at least some aspects of swarm cell differentiation might be involved in the ability of S. enterica var. Enteritidis to contaminate eggs. However, evidence against an association between swarm cell differentiation and egg contamination exists. S. enterica var. Pullorum efficiently contaminates eggs while S. enterica var. Enteritidis does so sporadically (Snoeyenbos, 1991, supra; Shivaprasad et al, Avian Dis., Volume 34, 548-557, 1990; Humphrey et al, Epidemiol. Infect., Volume 106, 489-496, 1991; Keller et al, Infec. Immun., Volume 63, 2442-2449, 1995). Since S. enterica var. Pullorum is historically aflagellate it was considered that either a) the ability to flagellate and undergo swarm cell differentiation was not involved in establishing invasive infections, or b) cellular differentiation of S. enterica var. Pullorum was inhibited.
U.S. Pat. No. 5,510,241 (Thorns) discloses that Salmonella microorganisms produce fimbrial antigens when they grow in vivo, e.g. in the gut, in animal tissues or fluids, in food products and in some natural environments but many of the fimbral antigens are not produced when they are grown in vitro on most culture media. The patent also discloses a medium for inducing Salmonella, such as for example S. enteritidis and S. dublin, to produce a specific fimbrial antigen during in vitro culture so that fast and very specific assays can be performed. The media identified by the Thorns patent, useful for inducing a specific fimbrial antigen, SEFA, on serotypes S. enteritidis and S. dublin, are media having at least 20% by weight of their nutrient composition made up of "defined" nutrients which are inorganic salts and/or organic compounds of known molecular structure. Peptone water and Enriched E broth are given as examples of preferred liquid media, with Slanetz broth, Heart infusion broth and Vogel Bonner broth as media capable of supporting expression of the specific antigen. The patent discloses, as examples of solid media, desoxycholate citrate agar, McConkey agar, Nutrient agar, Salmonella Shigella agar, Sheep blood agar and Xylose Lysine descholate. Oxoid Isosensitest and Sensitest agars are disclosed as those which are more potent in supporting the expression of the antigen.
Existing medias do not supply bacteria with essential ingredients that enable them to display a full range of potential physiological behavior in vitro. There remains a need in the art for more effective media which maintains cell viability and induces differentiation. The present invention described below is a growth supplement for media which induces and/or maintains differentiation and maintains viability of cells, especially bacterial cells and is different from prior art media.